The invention is related to the area of genome analysis. In particular it is related to the field of identification of genotypes.
Obtaining genotype information on thousands of polymorphisms in a highly parallel fashion is becoming an increasingly important task in mapping disease loci, in identifying quantitative trait loci, in diagnosing tumor loss of heterozygosity, and in performing association studies. A currently available method for simultaneously evaluating large numbers of genetic polymorphisms involves hybridization to allele-specific probes on high density oligonucleotide arrays. In order to practice that method, redundant sets of hybridization probes, typically twenty or more, are used to score each allelic marker. A high degree of redundancy is required to reduce noise and achieve an acceptable level of accuracy. Even this level of redundancy is insufficient to unambiguously score heterozygotes or to quantitatively determine allele frequency in a population. Because of these limitations, there is a need in the art for more reliable and more quantitative methods to perform genomic analysis at polymorphic loci.
The technique of allele-specific polymerase chain reaction (ASPCR) can be applied to allele identification and quantitative analysis of allele frequency. However, this technique suffers from cross reactivity between amplified products when hybridizing to probes which differ by only a single nucleotide base. A partial solution to the cross-reactivity problem has been achieved by the addition of sequence tags to the ASPCR primers. The incorporation of tags in ASPCR primers can itself interfere with the identification of the amplification products because unreacted primers or partially extended products can compete with full products for hybridization to the probes. Thus, there is a further need in the art for methods and materials which permit the use of tags in the analysis of polymorphic loci without interference from incompletely reacted products.
It is an object of the invention to provide methods and compositions for the identification of nucleotides at a polymorphic locus in a nucleic acid sequence. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a method for determining a nucleotide at a polymorphic locus. A region of double stranded DNA comprising a polymorphic locus is amplified to form an amplified DNA product using a pair of primers. A first primer of the pair terminates at its 3xe2x80x2 end at the polymorphic locus. The first primer also comprises a 3xe2x80x2 portion which is complementary to the region of double stranded DNA and a 5xe2x80x2 portion which comprises the same sequence as all or a portion of a probe on a solid support which is not complementary to the region of double stranded DNA. The amplified DNA products are labeled to form labeled amplified DNA products. The labeled amplified DNA products are hybridized to the probe on the solid support.
Another embodiment of the invention provides a pair of primers which specifically amplify an allelic form of a polymorphic locus. A first primer of the pair comprises a 3xe2x80x2 portion which is complementary to a region of DNA comprising the polymorphic locus. The first primer also comprises a 5xe2x80x2 portion which comprises the same sequence as all or part of a probe on a solid support. The sequence is not complementary to the region of DNA comprising the polymorphic locus. The first primer terminates in a 3xe2x80x2 nucleotide which is complementary to a distinct allelic form of the polymorphic locus.
The invention thus provides the art with sensitive and specific methods and compositions for identification of polymorphic nucleotides in a DNA sample which may be from one or more individuals.